The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) and N'-nitrosonornicotine (NNN) are likely to play a major role in cancers of the lung, oral cavity, esophagus, and pancreas caused by tobacco products. The goal of this proposal is to establish methodology which will enable one to understand the metabolic activation of NNK and NNN in humans, and its relationship to carcinogenesis. The hypothesis being tested is that levels of adduct formation are related to cancer susceptibility. The specific aims are: 91) Develop a combined GC-MS method to quantify hemoglobin adducts of NNK/NNN, polynuclear aromatic hydrocarbons (PAH), and aromatic amines in humans. This methodology will allow assessment of the relative levels of these carcinogen adducts in a given person's blood, and will thereby provide insights on their mechanisms of activation and roles in tobacco-related cancers. (2) Improve the present methodology for quantifying NNK/NNN hemoglobin and DNA adducts in humans by a) developing new internal standards based on modified hemoglobin or DNA; b) streamlining the methods; c) decreasing background; and d) increasing sensitivity. (3) Determine levels of human lung microsomal alpha-hydroxylation of NNK at its methylene and methyl carbons, and compare to levels of NNK/NNN hemoglobin adducts in the same individuals. Investigate the relationship between urinary 3-methyladenine and NNK/NNN hemoglobin adducts in smokers on controlled diets. (4) Develop a method to assay NNAL-glucuronide, a newly discovered NNK metabolite, in human blood and urine. (5) Obtain structural information on the unknown adducts seen in 32p-postlabelling analyses of DNA from lung tissue from smokers. The results of these studies will provide important new insights on mechanisms of cancer induction in humans.